JPS58165794A - Production of unsaturated dicarboxylic acid utilizing microorganism - Google Patents

Abstract

PURPOSE:A specific microorganism in Candida is made to act on a specific straight-chain unsaturated fatty acid or its alkyl ester aerobically to produce an unsaturated dicarboxylic acid in high yield. CONSTITUTION:As the microorganism, is used Candida tropicalis 1098, MD-105 or BR-254, while the straight-chain unsaturated fatty acid or its alkyl ester has a structure of formulaI(m, n are 0 or positive integer where m+n>=6). The strain is directly inoculated to the culture medium containing the straight-chain unsaturated fatty acid or its alkyl ester. Or the microorganism is previously cultured in a medium containing a carbon source that the microorganism can assimilate and the resultant cell bodies are made to react with the unsaturated acid or its ester in a medium containing a phosphate buffer solution.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing α,ω-linear unsaturated dicarboxylic acids from linear unsaturated fatty acids or alkyl esters thereof using microorganisms.

Conventionally, there has been a method for producing α,ω-linear saturated dicarboxylic acids from straight chain saturated fatty acids or their alkyl esters using microorganisms (for example, Japanese Patent Publication No. 38-1560; Japanese Patent Publication No. 5
0-19630. Japanese Patent Publication No. 53-25032) is known to directly produce α, ω-linear unsaturated fatty acids or their alkyl esters using microorganisms while retaining the unsaturated bonds of the compound. There are no known methods for producing dicarboxylic acids.

These α, ω-linear unsaturated fatty acids are, for example, Royal Jelly W (HOOC(CHs)s CH=CH-C0
0H) and traumatic acid (HOOC-(Cuma)a-CH
Although some compounds, such as ＝CH-COOH), have unique physiological activities, in general, there are heavy compounds that can be derived into various useful compounds by utilizing the characteristics of highly reactive unsaturated bonds. It is a complex compound. In addition, there are many naturally occurring unsaturated fatty acids that are cis-geometric isomers, such as cis-unsaturated dicarboxylic acids obtained by oxidizing the methyl group of oleic acid, which are often used as fragrances when heavily loaded with civetone. Although it is a heavy intermediate for synthesis, it is not easy to obtain such a cis-unsaturated dicarboxylic acid using conventional chemical synthesis methods.

The present invention has been developed in view of the above-mentioned current situation, and uses microorganisms to directly convert linear unsaturated dicarboxylic acids or alkyl esters thereof into α,ω-linear chains. An object of the present invention is to provide a method for producing an unsaturated dicarboxylic acid. The details of the present invention will be claimed below.

The main feature of the present invention is that a microorganism having the ability to convert the alkyl ester of Candida (Genus) into the corresponding linear unsaturated dicarboxylic acid is represents an integer of mtenn≧6
It is. ) is cultured aerobically in a medium containing a linear unsaturated fatty acid or an alkyl ester thereof, or by culturing the bacteria in advance in a medium containing another carbon source and combining the resulting bacterial cells with the linear unsaturated fatty acid or alkyl ester thereof. The point is that saturated fatty acids or alkyl esters thereof are reacted.

The microorganism used in the present invention is Candida caris B.
R-254 (FERMP4604) dH to 0 or less Ki, 1 Yandida tropicalis MD-105 (BP -10
0).

(1) Microscopic findings: Cell size and shape: short oval, 4-8μ
×5 to 11μ (2) Observations on the medium Shape on Nigelcose-Yeast Extract-Peptone-Agar medium: White to cream in color, soft and smooth.

(3) Maximum growth temperature lf: 41℃
~44℃ (4) Fermentable sugars Nigelcose + Lactose - Galactose + Melibiose - Sucrose
＋ Raffinose − Maltose ＋
meletitose - 74biose - inulin - trehalose + (5) Assimilation of carbon compounds nigercose + meletitose + galactose + inulin - D-lipose - soluble starch - rhamnose -
D-xylose + L-sorbose + L-
Arabinose + Sucrose + D-arabinose - Maltose + Ethanol
+Trehalose + Glycerol +Lactose - Erythritol -Melibiose - Ribitol + Raffinose
- Galactiol - D-Mannitol +
Salicin + D-glucitol +
DL-lactic acid 10 succinic acid + inositol -α-methyl-D-
61KNO, assimilation ability: None 7) Vitamin requirement: Biotin f8) Growth in hiJmin-deficient medium (9) Salt tolerance: 11-13% W/V 01 Guanosine-cytosine content 2 35.3% On the other hand, the linear unsaturated fatty acid used in the present invention is elaidic acid (trans-9-octadecenoic acid).

Oleic acid (cts-9-octadecenoic acid), petroselic acid (aim-6-octadecenoic acid), vaccenic acid (trans-11-octadecenoic acid), 2-decenoic acid, 2-undecenoic acid, 2-hexadecenoic acid, etc. I can give an example. Some of these fatty acids are solid under culture or reaction conditions, and in these cases it is also possible to use alkyl esters of the fatty acids to facilitate the reaction.

In the present invention, cells of the strain to be used are inoculated and cultured in a medium containing these linear unsaturated fatty acids or their alkyl esters, or a carbon source (for example, sucrose) that can be assimilated by the strain to be used is used.

Either of the so-called resting bacterial cell reactions is used, in which bacterial cells obtained by culturing in advance in a medium containing (glucose, molasses) are brought into contact with nuclear unsaturated fatty acids or their alkyl esters in a medium such as a phosphate buffer. Is possible. As a culture (or reaction) medium, it is sufficient as long as the strain used has good growth, and the addition of special ingredients is not required. usually,
1 to 40% by volume (or weight%) of unsaturated fatty acids or alkyl esters thereof are added to the medium, and the temperature is 25 to 40% by volume.
It is desirable to perform the culture (or reaction) at 35° C. and pH 6.0 to 7.5, and to supply sufficient oxygen during the culture.

After culturing (or reacting) as described above, adding sodium hydroxide or the like to the culture solution (or reaction product solution) to neutralize and dissolve the produced unsaturated dicarboxylic acid crystals,
When the sterilizing solution is separated and sulfuric acid, etc. is added to the resulting disinfectant solution, crystals of the product will precipitate. These product crystals are normal P3N! Although it can be easily recovered by J operation, it is also possible to recover it by means such as solvent extraction.

EXAMPLES The present invention will be described in more detail below with reference to Examples.

Example 1 30°C on malt extract agar slants.

Candida φ tropicalis MD-1 cultured for 24 hours
Three loopfuls of 05 (BP-100) cells were inoculated into a 500 d Erlenmeyer flask into which a 50-ml medium with the composition shown in Table 1 had been dispensed, and incubated at 30°C for 24 hours.
A seed culture solution was prepared by performing rotary shaking culture at a speed of rpm.

Table-1 Sucrose 30L-Asparagine 6g KH*POa 39 and HPO41
3.5g MdaSO4・7Hz0 10daMnSO4'5H108mp ZnSOa"7H 0 8m
9 Biotin 5μ yeast extract 1g The above composition was dissolved in 1 t of distilled water, and the pH of the medium was adjusted to 65.

Dispense medium 20- with the composition shown in Table-2 into a 500-capacity shoulder flask, add 2.0 g of elaidic acid (tran@-9-octadecenoic acid), and steam sterilize at 120°C for 15 minutes to sterilize. After completion of the cultivation, each flask was inoculated with 1 volume of the above seed culture solution, and cultured with reciprocating shaking at 30° C. for 96 hours at a speed of 155 reciprocations per minute.

Table-2 L-asparagine 6g KH* P 04 2.79, HPo
, 13.99M chiso, 7H elephant Q
O,6gFaSO4'7H*0 10
mgMnSO, 5I (*0 8 TQZ
n S O4” 7 H*0 8 11Q biotin 5 μl yeast extract 1 g The above composition was dissolved in 1 t of distilled water, and the pH of the medium was adjusted to 7.5.
Adjusted to.

Potash hydroxide grains were added to the culture solution obtained as described above to adjust the pH to 10 and dissolve fatty acids.
was collected. The internal standard Pal in the collected sample was 51 nl of an ether solution of tinic acid (67 nl as palmitic acid concentration).
ml) was added thereto, followed by acid precipitation with 2N hydrochloric acid, followed by extraction with diethyl ether. The ether extract was methyl esterified with diazomethane and analyzed using gas chromatography equipped with a flame ionization detector. As a result of analysis of the relative retention time of the product on the gas chromatogram (the gas chromatogram is shown in attached Figure 1) and the mass spectrometry spectrum of the product peak (shown in attached Figure 2), the product is It was identified as trans-9-octadeceneniic acid.

The concentrations of the product and unreacted elaidic acid in the culture solution were 13.9 g/l and 57.4 g/l, respectively.

Example 2 A seed culture solution prepared in the same manner as described in Example 1 was centrifuged at 8.000 rprrl for 5 minutes at 5°C. Wash with 0. The bacterial cell concentration in 1M phosphate buffer is 5g/l.
It was suspended so that Upper bacterial suspension 20-500-
Place in a shoulder flask and add 2,09% of elaidic acid.
and 30°C for 96 hours.

Reciprocal shaking was performed at a speed of 155 reciprocations per minute. As a result of analyzing the obtained reaction solution in the same manner as described in Example 1, it was found that the reaction solution contained 32.9q7t and 4t of trans-9-octadecenoic acid and unreacted elaidic acid, respectively.
7.89/ was found to exist.

Example 3 Example 1 except that petroselic acid (aim-6-octadecenoic acid) was used instead of elaidic acid as the culture substrate.
Culture was carried out in the same manner as described. The concentrations of cls-6-octadecenoic acid and unreacted petroselinic acid in the resulting culture were 5368 μl/l and s, respectively.
It was sg/4.

Example 4 Oleic acid (c
Culture was carried out in the same manner as described in Example 1, except that 1g-9-octadecenoic acid) was used.

The concentrations of c14-9-octadeceneniic acid and unreacted oleic acid in the obtained culture solution were 48.1 g/l and 15.4 g/l, respectively.

Example 5 Cultures were carried out in the same manner as in Example 2 using each of 2-decenoic acid, 2-undecenoic acid, 2-tridecenoic acid, and 2-hexadenoic acid as culture substrates. Unsaturated dicarboxylic acids corresponding to unsaturated fatty acids were produced at the concentrations shown below.

Example 6 Using the same procedure as in Example 1, oleic acid methyl ester,
Each of oleate ethyl ester, oleate isopropyl ester, and oleate butyl ester was used as a culture substrate (
When culturing was carried out with the amount added (2-) per flask, cis-9-octadeceneniic acid was produced from each substrate at the concentrations shown below.

For oleic acid methyl ester: 30.4g/l
tl- of oleic acid ethyl ester: 32.1 q
/l・For oleic acid isopropyl ester:
For 2B, 79/l oleic acid butyl ester:
31.59//! .

[Brief explanation of the drawing]

The attached FIG. 1 shows a gas chromatogram of ether extraction 11 of the culture obtained in Example 1. In the figure, peak A is methyl ester of valmitic acid added as an internal standard, peak B is methyl ester of elaidic acid, and peak C
is the product dimethyl ester of octadecenoic acid. FIG. 2 shows a mass spectrometry spectrum of a peak (peak C) corresponding to the product on the gas chromatogram shown in FIG. The numbers on the horizontal axis in the figure represent the mass number, and the vertical axis represents the relative intensity of the peak. 15-

Claims (1)

[Scope of Claims] A microorganism belonging to the genus Candida that has the ability to convert linear unsaturated fatty acids into corresponding linear unsaturated dicarboxylic acids is defined by the general formula % formula % (1) (where m and n are Hail zero or positive integer, m+n≧
It is 6. ) is cultured aerobically in a medium containing a linear unsaturated fatty acid or its alkyl ester (alkyl group has 1 to 4 carbon atoms), or the above bacteria are cultured aerobically in a medium containing a carbon source that can be assimilated by the bacteria. Reacting the bacterial cells obtained by culturing in advance in a culture medium with the above-mentioned linear unsaturated fatty acid or its alkyl ester to produce and collect α, ω-linear unsaturated dicarboxylic acid corresponding to the unsaturated fatty acid. α characterized by
. Method for producing ω-linear unsaturated dicarboxylic acid.